Targeted delivery of bioaffecting compounds for the treatment of cancer

ABSTRACT

A homogeneous conjugate for targeting and treating diseased cells wherein the conjugate comprises an anti-cancer drug and a targeting protein, wherein said anti-cancer drug is selected from the group consisting of heat sensitizers, photosensitizers and apoptosis inducing compounds, a method for making such a conjugate, and methods for using the conjugate. The targeting protein is preferably transferrin.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 USC § 371 National Phase Entry Application from PCT/US02/11890, filed May 15, 2002, and designating the U.S, which claims priority benefit of U.S. Provisional Application Nos. 60/290,681 filed May 15, 2001, 60/291,016 filed May 16, 2001 and 60/291,015 filed May 16, 2001. The disclosure of the International Application and the three U.S. Provisional Applications are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of bio-affecting materials useful in the treatment of diseased cells, such as cancer cells and, more specifically to a conjugate of a targeting agent and a bio-affecting material, where the bio-affecting material is a heat sensitizer, a photosensitizer or an apoptosis inducing compound.

BACKGROUND OF THE INVENTION

Two of the most devastating problems in cancer treatment are drug-toxicity, which debilitates patients, and drug-resistance, which is normally countered with even higher drug dosages and thus amplifies the problem of drug-toxicity, often resulting in death. One way to solve the problem of drug-toxicity is to target drugs for delivery only to cancer cells. Many researchers are working to develop antibodies to deliver drugs to targeted cells, and this approach holds promise, but antibodies are not without problems. For example, antibodies often bind to normal tissues, and they also can damage blood vessels (e.g., vascular leak syndrome) and cause dangerous allergic reactions (e.g. anaphylaxis).

Research is also progressing in connection with the use of conjugates of transferrin and doxorubicin, daunomycin, methotrexate, vincristin, 6-mercaptopurine, cytosine arabinoside, cyclophosphamide, and radioiodine as described in U.S. Pat. Nos. 5,108,987; 5,000,935; 4,895,714; and 4,886,780. The inventions described in these patents does not use antibodies. Instead, it uses a protein found in normal human blood. This protein is transferrin, which delivers iron. Normal cells rarely require iron, but cancer cells require large amounts of iron to maintain their pathologically increased rates of metabolism. Because cancer cells require more iron, they have transferrin receptors substantially permanently on their surfaces, whereas normal cells do not. These inventions exploit these receptors by administering anticancer drugs conjugated with transferrin, which delivers the drugs substantially only to the surface of cancer cells.

Drug targeting spares normal cells, requires less drug, and significantly diminishes drug-toxicity. In contrast, when anticancer drugs are administered without being targeted, they kill normal cells as well as cancer cells. They are particularly toxic to cells of the immune system and to the system responsible for blood clotting. Thus, infections and bleeding are principal complications of chemotherapy in cancer patients. These complications require expensive services, hospitalizations, intensive care, and life-support systems, which are uncomfortable and expensive for the patient. These problems are largely preventable by using targeted delivery systems.

The problem of drug-toxicity consumes huge blocks of doctors' and nurses' time, and is responsible for much of the cost of cancer care. For example, it is commonly understood that about 70% of calls to oncologists relate to a problem of drug-toxicity. Today there is no satisfactory way to treat drug-toxicity, except to use less drug. In the absence of targeted delivery the use of less drug is counterintuitive in the case of drug resistant cancers. Targeted delivery allows the use of less drug, because more of the administered drug is delivered specifically to cancer cells rather than being nonspecifically distributed around the body. In this sense, targeted delivery is like shooting with a rifle, while conventional delivery is like shooting with a shotgun. A solution to the problem of drug-toxicity will dramatically transform chemotherapy in cancer patients. It is a purpose of this invention to reduce such adverse effects of chemotherapy.

The problem of drug-resistance is equally as serious as the problem of drug-toxicity. This problem is typified by a patient diagnosed with cancer who is treated and responds with a symptomless remission that lasts many months, and who later sees the cancer returns in a form that no longer responds to any known drug. This scenario of drug-resistance is all too common, Yet today there is no satisfactory solution, except the use of larger amounts of more powerful drugs, that in turn can cause serious drug-toxicity problems, often resulting in death. Thus, a solution to the problem of drug-resistance would significantly diminish the problem of drug-toxicity. A major effort was devoted to the use of P-glycoprotein inhibitors such as verapamil (Ford, Hematol/OncolClin N Am 1995; 9:337), cyclosporine (Bartlett et al, J Clin Oncol 1994; 12:835) and cyclosporine derivatives such as SDZ PSC 833 (Kusunoki et al., Jpn J Cancer Res 1999; 89:1220), and tamoxifen (Pommerenke et al., J Cancer Res Clin Oncol 1994; 120:422), but these approaches have not been clinically satisfactory because they introduced new problems in the pharmacokinetics of chemotherapy (Sikic et al., Cancer Chemother Pharmacol 1997; 40:S13). Other approaches are the design of MDR-reversing drugs (Naito & Tsuruo, Cancer Chemother Pharmacol 1997; 40:S20); the use of P-glycoprotein antisense oligonucleotides (Bertram et al, Anti-Cancer Drugs 1995; 6:124); the use of retrovirus-mediated transfer of anti-MDR ribozymes (Wang et al., Human Gene Therapy, 1999; 10:1185), and the design of chemotherapy drugs that are not removed from cancer cells by MDR or MRP pumps (Mankhetkom et al., Mol Pharmacol 1996; 49:532), but none of this research has provided a solution to the clinical problem of drug resistance (Arceci, Br J Haematol 2000; 110:285). Another approach is to circumvent the MDR/MRP pumps by delivering anti-cancer drugs as conjugates of larger molecules such as albumin (Ohkawa et al., Cancer Res 1993; 53:4238), alpha-fetoprotein (Moskaleva et al., Cell Biol Int 1997; 21:793) and dextran (Fong et al., Anticancer Res 1996; 16:3773). Although these are variously effective at evading experimental drug resistance, they are unproven in patients. Protein-targeted drug delivery can overcome the problem of drug-resistance. Thus, another purpose of the present invention is to resolve the issue of painful and expensive deaths from drug-resistant cancers.

The effectiveness of proteins conjugated with bio-affecting molecules has been demonstrated and is described in the U.S. patents mentioned above. It has been determined; however, that the efficiency of such conjugates in treating stressed cells, such as cancer cells, is reduced by the presence of agglutinated conjugates or by the presence of conjugates of a bio-affecting molecule with protein fragments or with two or three protein molecules and is greatly enhanced when the protein to bio-affecting molecule ratio is closer to 1:1 or 1:2, depending on the bio-affecting molecule. Obtaining conjugates of higher efficiency has, in the past, been a slow, tedious and expensive process that requires separating a fraction of conjugate having the desired average ratio of bio-affecting molecule to protein from a larger sample comprising such molecules conjugated with protein fragments, with a plurality of proteins and proteins conjugated with a plurality of bio-affecting molecules. Using homogeneous protein-drug conjugates in which the protein component carries a predetermined number of bio-affecting molecules can more effectively kill both drug-resistant and drug-sensitive cancer cells. In the past the expense and inefficiency inherent in producing useful conjugates in a useful volume has been a problem for the commercialization of such conjugates and for their widespread use in medicine. There is a need for a substantially homogeneous drug-protein conjugate and for a method of making such a conjugate that is more efficient, more precise and less costly. It is one purpose of this invention to provide such a homogeneous conjugate made by a more efficient method.

DESCRIPTION OF THE RELATED ART

The first report of transferrin receptors on human cancer cells was by Faulk and colleagues in 1980 (1). This was followed by many reports of transferrin receptors in different types of human cancers (2), as seen in the following Table.

Tumor Studied References Breast  1, 3 Leukemia  4, 5 Lung  6 Brain  7 Liver  8 Bladder  9 Gastrointestinal 10 Ovary 11 Non-Hodgkin's lymphoma 12 Lymphoma/melanoma 13, 14 Nasopharyngeal 15 Cervix 16 Transferrin Receptors on Normal and on Cancer Cells.

No single study has asked if all human cancers have up-regulated transferrin receptors, or if all normal cells have down-regulated transferrin receptors, but data from many quarters suggest that the answer to both questions is yes. For example, immature erythrocytes (i.e., normoblasts and reticulocytes) have transferrin receptors on their surfaces, but mature erythrocytes do not (17). Circulating monocytes also do not have up-regulated transferrin receptors (8), and macrophages, including Kupffer cells, acquire most of their iron by a transfenrin-independent method of erythrophagocytosis (19). In fact, in vivo studies indicate that virtually no iron enters the reticuloendothelial system from plasma transferrin (for review, see reference 20). Macrophage transferrin receptors are down regulated by cytokines such as gamma interferon (21), presumably as a mechanism of iron-restriction to kill intracellular parasites (22).

In resting lymphocytes, not only are transferrin receptors down regulated, but the gene for transferrin receptor is not measurable (23). In contrast stimulated lymphocytes up regulate transferrin receptors in late G₁ (24). Receptor expression occurs subsequent to expression of the c-myc proto-oncogene and following up-regulation of IL-2 receptor (25), and is accompanied by a measurable increase in iron-regulatory protein binding activity (26), which stabilizes transferrin receptor mRNA (27). This is true for both T and B lymphocytes (28), and is an IL-2-dependent response (29).

Cell stimulation resulting in the up regulation of receptors for transferrin is known to result from stress experienced, for example, by cells invaded by a viral or protozoan factor and by cancer cells.

Up-and-down regulation of transferrin receptors for normal and tumor cells has been shown by studies of antigen or lectin stimulation (i.e., receptor up-regulation), and by studies of differentiation models (30–33) using retinoic acid (i.e., receptor down-regulation). Base-line data from these experimental models suggest that these receptors are down-regulated from the plasma membranes of most normal, adult, resting human cells (34). Exceptions are the circulatory barrier systems, which include the materno-fetal barrier with its transferrin receptor-rich syncytiotrophoblast (35); the blood-brain barrier with its transferrin receptor-rich capillary endothelial cells (36); and, the blood-testis barrier with its transferrin receptor-rich Sertoli cells (37).

Transferrin-Drug Conjugates in Laboratory Animals

The efficacy of transferrin-drug conjugates has been investigated in several animal models. For example, conjugates of transferrin with diphtheria toxin decreased xenografted gliomaas in nude mice by 95% on day 14, and the gliomas did not recur by day 30 (74). Also, glutaraldehyde-prepared transferrin-doxorubicin conjugates have been found to rescue nude mice from death by human mesothelioma cells, significantly prolonging life compared to animals treated only with doxorubicin (75). In addition, transferrin has been coupled to herpes simplex thymidine kinase by using biotin-streptavidin technology, and these conjugates significantly prolonged life in nude mice inoculated with metastasizing K562 tumor cells (76). Finally, the maximum tolerated dose of human transferrin-doxorubicin conjugates in nude mice has been found to be 20 mg/kg (iv) for conjugates and only 8 mg/kg (iv) for free drug (41).

Transferrin-Drug Conjugates in Human Patients

There are two clinical reports of transferrin-drug conjugates. The first, published in 1990, was a preliminary study of seven acute leukemia patients treated intravenously with 1 mg/day of glutaraldehyde-prepared transferrin-doxorubicin conjugates for 5 days. With these low doses, there were no toxic effects and the number of leukemic cells in peripheral blood of the 7 patients decreased by 86% within 10-days following therapy (77). In addition, there was no extension of disease as assessed by examination of bone marrow biopsies before and after treatment.

The second, published from the NIH in 1997, involved 15 patients with recurrent brain cancers treated with thioether-bonded transferrin conjugates of a genetic mutant of diphtheria toxin (44). The conjugates were delivered by high-flow interstitial microinfusion, which has been shown to produce effective pefusion of radiolabeled transferrin in primate brains with minimal inflammatory responses (78). Magnetic resonance imaging revealed at least a 50% reduction in tumor volume in 9 of the 15 patients, including 2 cases of complete remission (44).

There is an unpublished clinical study of 23 patients with advanced ovarian cancer who were randomized into test (12 patients) and placebo (11 patients) groups. The test group received transferrin-doxorubicin conjugates equivalent to 1 mg doxorubicin per day on days 15 through 19 of monthly treatment cycles. A significant difference was revealed by Cox regression estimates of survival rates for patients treated with transferrin-doxorubicin conjugates when the time between diagnosis and randomization was 18 months.

Another unpublished study is a 22-year old male with metastatic disease from a sarcoma of his right atriunm who was treated by conventional protocols without response. His lungs were filled with metastatic lesions when his physician father obtained an IND from the FDA for the use of transferrin-doxorubicin conjugates, and treatment was begun in August, 2000. By November, the lungs were substantially cleared of metastatic lesions, and by January there was no radiological evidence of tumor. He presently (May 2002) is active, receiving only transferrin-doxorubicin.

The targeted delivery of drugs has the advantage of increasing efficacy while using less drug, thereby decreasing toxicity and causing less damage to normal cells, all of which effectively decrease costs and increase the quality of patient care. Targeted delivery also avoids drug-resistance, which is activated by the non-specific entrance of drugs into cells (79). Because transferrin-drug conjugates can enter cells specifically by employing a receptor-specific pathway (80,81), they are trafficked around drug-resistance mechanisms, such as efflux pumps in resistant cells.

It was reported in 1992 that transferrin-doxorubicin conjugates effectively kill multi-drug resistant cells (82). This finding was confirmed in 1993 (83), and was extended to several types of drug-resistant cells in 1994 (84), 1996 (85) and 2000 (86). Interestingly, doxorubicin immobilized on solid carriers also has been shown to be effective against doxorubicin-resistant cells (60,87). In fact, a concept is emerging that vectorization of doxorubicin with one of several peptide vectors is effective in overcoming multi-drug resistance (88).

Preparation of Transferrin-Drug Conjugates.

A method for the preparation of transferrin-doxorubicin conjugates was published first m 1984 (38), following which there have been many reports of methods for the preparation of transferrin-drug conjugates, some of which are listed in the following Table.

Transferrin Label Method Used References Doxorubicin Glutaraldehyde 38, 39, 40 Doxorubicin Maleimide 41 Mitomycin C Glutaryl Spacer 42 Neocarzinostatin Succinimide 43 Diphtheria Toxin Thioester 44 Chlorambucil Maleimide 45 Paclitaxol Glutaraldehyde 46 Daunorubicin Glutaraldehyde 47 Titanium Carbonate 48 Insulin Disulfide 49 Gallium Carbonate 50 Platinum Methionine 51 Saporin/ricin Succinimide 52 Ruthenium Bicarbonate 53 Growth Factor Fusion Protein 54 HIV Protease Recombinant 55

Transferrin conjugates of doxorubicin have been prepared by using glutaraldehyde-mediated Schiff base formation (56,57), which forms an acid-resistant bond between epsilon-amino lysine groups of transferrin and the 3′amino position of doxorubicin Such conjugates of doxorubicin can kill cancer cells through a plasma membrane-mediated mechanisms (for review, see reference 58). Although DNA intercalation is an established mechanism of cell death by doxorubicin, immobilized doxorubicin corrugated with proteins capable of binding with receptors on the cell surface activate plasma membrane-mediated mechanisms to kill cells (59,60). It thus appears that conjugates of doxorubicin with transferrin kill cells by activating plasma membrane-mediated mechanisms that involve both doxorubicin and transferrin receptors.

SUMMARY OF THE INVENTION

It is apparent that targeting agent-drug conjugates have a generic possibility of changing how drugs are delivered, as well as a specific possibility of changing how drugs are delivered to cancer patients. In the present invention, non-antibody proteins such as transferrin, ceruloplasmin, vitamin binding proteins such as transcobalamin, hormones, somatostatin, cytokines, low density lipoproteins, growth factors, growth factor-like molecules, are used as targeting agents in combination with anti-cancer compounds. Because transferrin and transcobalamin are excellent carriers for delivering compounds into drug-sensitive and drug resistant cancer cells, the targeted delivery system in the present invention preferably consists of transferrin or transcobalamin coupled to anticancer compounds selected from the group consisting of heat sensitizers; photosensitizers and apoptosis inducing confounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to conjugates with a targeting agent and an antitumor agent. The targeting agents according to the present invention include butane not limited to transferrin, ceruloplasmin, somatostatin, vitamins, vitamin binding proteins such as transcobalamin, hormones, cytokines, low density lipoproteins, growth factor-like molecules, folic acid-like molecules and growth factors. The anti-tumor agents include but are not limited to heat sensitizers such as hematophorphyrine and low-dose verapamil, apoptosis inducing compounds such as deferoxamine, and photosensiers such as porfimer sodium, metatetrahydroxyphenylchlorin, and hematophorphyrin derivatives. Preferably the targeting agent is transferrin or transcobalamin. Preferably the anti-tumor agent and the targeting agent are conjugated by means of a linker. Suitable linkers include but are not limited to glutaraldehyde (Bérczi et al., Arch Biochem Biophys 1993; 300: 356), disulfide coupling (Sash et al., Jap J Cancer Res 1993; 84: 191) and benzoyl hydrazone (Kratz et al., J Pharm Sci 1998; 87: 338).

Transferrin-Heat Sensitizer Conjugates

Hyperthermia is widely used in cancer treatment either alone or in combination with chemotherapy and radiotherapy (Kong et al., Int J Hyperthermia 1999; 15: 345; Raaphorst et al., Oncology Reports 1998; 5: 971; Mager et al., Anticancer Res 1999; 19: 3403). In combination treatments, hyperthermia (41–45° C.) has been shown to be an effective radiation sensitizer (Sukurai et al., Int J Rad Biol 1999; 75: 739; Harima et al., Cancer 2000; 88: 132). Heat enhances the cytotoxic effect of certain anticancer agents such as bleomycin (Khadir et al., Arch Biochem Biophys 1999; 370: 163), carboplatin (Steller et al., Cancer Chemother Pharmacol 1999; 43: 106), doxorubicin (Kawai et al., Cancer 1997; 79: 214) and cisplatin(Raaphoist etal., Oncol Reports 1998; 5: 971; Benoit et al., Chirurgie 1999; 124:375). Hyperthermia is used in some therapy-resistant malignancies (Wessalowski et al., Int J Hyperthermia 1999; 15: 455) and high temperature hyperthermia (greater than 50° C.) has been used for selective tissue destruction as an alternative to conventional invasive surgery (Diederich et al., Ultrasound In Med Biol 1999; 25: 871).

The effectiveness of hyperthermia is strongly dependent on the ability to localize and maintain therapeutic temperature elevations (Diederich et al., Ultrasound In Med Biol. 1999; 25: 871). To achieve better results, several techniques have been used to produce controlled temperature increases in tissue. Some of these techniques involve the use of YAG Lasers (Graeber et al., Laryngoscopy 1999;109: 447), radiofrequences (Wust et al., Int J Hyperthermia 1998;14: 459), ultrasound (Diederich et al., Ultrasound In Med Biol 1999; 25: 87 I), and microwaves (Finger, Ophthalmol 1997;104:1794). Hyperthermia is more effective at high temperatures, but its use is limited by the need to protect healthy tissues from heat damage.

Heat induces toxic characteristics in several compounds that are not toxic at normal body temperature. An example of this is hematophorphyrine. At 41.5° C. this molecule is a potent heat (or hyperthermia) sensizer, and its ability to sensitize increases in a dose-dependent manner (Saito et al., Int J Hyperthermia 1998;14: 503). Another example of a heat (or hyperthermia) sensitizers is the use of low-dose verapamil at 42° C. (Shchepotin et al., Int J Hyperthermia 1997;13: 547). Heat sensitizers do not elevate local temperatures. They function by producing increased local toxicity in the presence of heat. If non-toxic compounds that produce toxicity at higher than body temperature, or toxic compounds that increase their toxicities at elevated temperature, could be delivered only to cancer cells, then hyperthermia could be used more efficiently to treat cancer. Such a targeted delivery system would not damage healthy tissues, which would receive only the effects of elevated temperatures.

This invention introduces a way of selectively delivering compounds that are highly toxic for cancer cells at increased temperatures. This targeted delivery system involves the use of transferrin as the carrier of the heat (or hyperthermia) sensitizer.

The use of targeted delivery of heat sensitizers would make the outcome of hyperthermia more effective by itself or in combination with other treatments, since heat sensitive toxicity can be concentrated in cancer cells. In this way, damage of healthy tissues is minimal, as most sensitizers can work at relatively low temperatures (e.g., 41–42° C.).

Since transferrin is an excellent carrier for delivering compounds to drug-sensitive and drug resistant cancer cells, the targeted delivery system in this invention consists of transferrin coupled to heat sensitizers. Transferrin for use in human patients is commercially available (for example, from the Finish Red Cross). Several coupling processes such as glutaraldehyde coupling (Bérczi et al., Arch Biochem Biophys 1993; 300: 356), disulfide coupling (Sash et al, Jap J Cancer Res 1993; 84: 191) and benzoyl hydrazone coupling (Kratz et al., J Pharm Sci 1998; 87: 338) have been used to couple transferrin with other compounds. The broad variety of coupling procedures allow the conjugation of a wide range of sensitizers to transferrin, resulting in either permanent or dissociable bonding of sensitizers with the transferrin molecule. Following the conjugation of transferrin to the selected heat sensitizer, the conjugates should be separated from the uncoupled components, for example by using chromatographic and/or dialysis techniques. Examples of suitable heat sensitizers include but are not limited to doxorubicin, cisplatin and hematophosphrine.

Transferrin-Photosensitizer Conjugates

Photochemotherapy of cancer often is called photodynamic therapy (PDT). This form of therapy arose in the late 1970s and appeared as an alternative form of treatment for localized inoperable lesions (Dougherty et al., Cancer Res 1978; 38:2628). It involves the administration a tumor-localizing agent (Hayata et al., Chest 1982; 81:269), which may require metabolic changes (e.g., a prodrug), followed by activation of the agent by light of a specific wavelength (Sharman et al., Meth in Enzymol 2000; 319:376). This results in production of a sequence of photochemical and photobiological reactions, such as the production of singlet oxygen (Yuan et al., Radiation Res 1997; 148:386) that initiate tumor necrosis (Ochsner, J Photochem Photobiol 1997, 39: 1) via thrombosis (Henderson & Dougherty, J Photochem Photobiol 1992; 55:145) and apoptosis (Luo & Kessel, Biochem Biophys Res Corn 1996; 221:72).

Progress in PDT during the 1990s has included development of more efficient photosensitizers that absorb at longer wavelengths and require less energy for activation, thus allowing deeper penetration. New fiber optics also have been developed that allow illumination of difficult geometric configurations, and smaller, less expensive and more mobile lasers have become more readily available. This research and development has allowed PDT to evolve into a more widely used therapeutic modality, both for the primary treatment of cancer and for an adjunct to surgical debulking of larger tumors

Treatment with PDT has been used in a wide variety of cancers, including basal cell carcinomas (Stefanidon et al., Euro J Dermatol 2000; 10:351), lymphomas (Orenstein et al., Dermatol Surg 2000; 26:765), stomach cancer (Gassner et al., Gastrointest Endoscop Clin N Am 2000; 10:461) and leukemia (Danilatou et al., Leukemia Res 2000; 24:427). Indeed, a recent review lists more than 60 types of cancer that have been treated with the FDA-approved photosensitizer Photofrin (porfimer sodium). In addition to its use in cancer treatment, PDT also has been used in Dermatology (Hongcharu et al., J Invest Dermatol 2000; 115:183), Ophthalmology Tannous et al, Invest Ophthal Visual Sci 2000; 41:749), and recently PDT has been used in angioplasty procedures (Rockson et al, Circulation 2000; 102:591).

The first photosensitizer to receive FDA approval was Photofrin, produced by QLT Photo Therapeutics in Canada Another widely studied photosensititer is aminolevulnic acid. Other molecules in clinical trials are SnET2, which is a chlorin photosensitizer produced as Purlytin by Pharmacia/Upjohn. Another molecule is mTHPC produced by Scotia Pharmaceuticals as Foscan, which is a metatetrahydroxyphenylchlorin. Macular degeneration has been inhibited by using the photosensitizer Verteporfin, which is a haematophorphyrin derivative. These and several other molecules used as photosensitizers have been reviewed by Stewart and colleagues (Radiother Oncol 1998; 48:233) and by Dougherty and colleagues (J Natl Cancer Inst 1998; 90:889).

Since the presence of transferrin receptors is restricted principally to the surface of tumor cells, as originally described by Faulk and colleagues in the Lancet (1980; 2:390), the use of transferrin as the carrier of photosensitizers virtually assures that the drug will be targeted only to cancer cells, thus allowing the effects of PDT to be localized to the tumor and only to the tumor. In addition to minimizing collateral damage of normal cells, targeting of the photosensitizer drugs as conjugates of transferrin allows the use of lower doses of drugs, which will diminish the side effects of some drugs. For example, solar photosensitivity is a dose-related problem encountered in some patients treated with Photoprin.

The present invention of targeting the delivery of photosensitizer drugs as conjugates of transferrin to transferrin receptors on the surface of cancer cells has several advantages that derive from the biology of the carrier. The foremost of these is the property of transferrin drug conjugates to cross the blood-brain-barrier (Broadwell et al., Exp Neurol 1996; 142:47). This property is endowed in the molecule by the normal presence of transferrin receptors on the luminal surface of endothelial cells that provide the microcirculation of the blood-brain-barrier (Moos & Morgan, Cell Mol Neurobiol 2000; 20:77). This has been utilized to deliver molecules from the blood circulation into the brain as conjugates of transferrin (Park et al., J Drug Target 1998; 6:53), which allows for the targeted use of PDT in patients with brain cancer. Another biological property of transferrin that positions it as an ideal carrier for photosensitizer drugs is that it is endocytosed into cells after being bound by transferrin receptors (Berczi et al, Eur J Biochem 1993; 213:427); This property can localize photosensitizers to the inside of cancer cells, which concentrates the drug and augments killing. In this regard, it is relevant that doxorubicin conjugates of transferrin have been used to kill doxorubicin-resistant cancer cells that were not killed by 10-fold higher concentrations of free doxombicin (Fritzer et al., Int J Cancer 1992; 52:619).

In the following discussion transferrin is illustrative of the targeting protein Transferrin can be isolated from normal blood plasma, purchased from any of several companies, including both the American and Finnish Red Cross, or be prepared from recombinant technology (Ali et al., J Biol Chem 1999; 274:24066). To form a drug:transferrin conjugate, the transferrin molecule must be modified in such a way as to prepare it to be coupled with a photosensitizer drug. For example, transferrin must be thiolated in order to form maleimide derivatives of drugs (Kratz et al., J Pharm Sci 1998; 87:338). The exact method of conjugation will depend upon the chemistry of the photosensitizer to be coupled with transferrin. For example, Protofrin is a polyporphrin containing ester and ether linkages, suggesting one type of carrier (Dubowchik & Walker, Pharmacol & Therap 1999; 83:67), other drugs contain NH2-groups that can be coupled to transferrin by using bifunctional reagents such as glutaraldehyde, and others require disulfide coupling (Sasaki et al., Jap J Cancer Res 1993; 84:191). The wide variety of coupling procedures allows the conjugation of a broad range of photosensitizer drugs with transferrin, resulting in either permanent or dissociable bonding of drugs with the transferrin molecule (Barabas et al., J Biol Chem 1992; 267:9437). Whatever procedure is used, after the coupling reaction the drug-protein conjugates must be separated from uncoupled drug and/or free protein, preferably by using chromatography or dialysis techniques.

Transferrin-Apoptosis Inducing Compound Conjugates

A mechanism for iron-restricted cytotoxicity in cancer is the potentiation of programmed cell death. Deferoxamine has been found to increase arabinoside-rnediated apoptosis of human myeloid leukemia cells (Leardi et al., Br J Haematol 1998; 102:746). There is considerable evidence that a major pathway for apoptosis involves calcium-mediated down-stream signaling subsequent to ligand-binding of programmed cell death surface receptor CD95 (Kass & Orrenius, Environ Health Perspec 1999; 107(Suppl 1):25). Similarly, the transferrin receptor functions as a signal-transduction molecule for its own recycling by increasing intracellular free calcium concentrations (Sainte-Marie et al., Europ J Biochem 1997; 250:689). Taken together, these apparently unrelated observations indicate a hitherto undescribed relationship between calcium, iron and transferrin receptors in drug resistant cancer cells.

In order to test the relationship between calcium and transferrin receptors, the effect of calcium channel inhibitors on the regulation of transfernin receptors was studied in drug-sensitive and drug-resistant cancer cells. The results of these experiments show that both organic (e.g. verapamil) and inorganic (CdCl₂) calcium channel blockers cause differential effects on the regulation of transfernin receptors. For example, both types of channel blockers initiated down-regulation of receptors, but more down-regulation was observed on drug-resistant cells than on drug-sensitive cells. Since the half-life of down-regulation was about eight hours, the decrease of surface receptors did not result from inhibition of exocytosis. The addition of free iron or transferrin-bound iron also had a differential effect on the down-regulation of transferrin receptors. The effect was similar but even more pronounced, namely iron down-regulated receptors much more on drug-resistant cells than on drug-sensitive cells, and produced about the same half-life as calcium channel blockers. The addition of both iron and calcium channel blockers had no effect on cell viability.

In light of the above findings, another series of experiments were done to determine whether the restriction of iron by deferoxamine had differential effects on drug-sensitive and drug-resistant cancer cells. The results of these experiments revealed that low concentrations of deferoxamine up-regulated transferrin receptors on both drug-sensitive and drug-resistant cells, but increasing concentrations of deferoxamine decreased transferrin receptor expression by drug-resistant cells, and viability experiments showed that the higher concentrations killed drug-resistant but not drug-sensitive cells; For example, after 48 hours in a given concentration of deferoxamine, drug-sensitive cells showed 93% viability, but only 16% of drug-resistant cells were alive. In summary, these three different types of experiments revealed a marked instability of transferrin receptor regulation in drug-resistant cells, and showed that iron depletion rapidly caused death of the drug-resistant cells.

The present invention is a way to target and kill drug-resistant cells in cancer patients. In a preferred embodiment, a conjugate of a drug carrier that targets an iron chelator to drug-resistant cancer cells is prepared. Transferrin is a normal blood protein that is well suited to deliver an iron chelator, because tumor cells have transferrin receptors on their surface and normal, adult, resting cells do not (Berczi et al., Arch Biochem Biophys 1993; 380:356). There are many iron chelators that can be conjugated with transferrin and delivered to drug-resistant cancer cells (Tsafack et al, Mol Pharmacol 1995; 47:403), but the chemistry of deferoxamine renders it well suited for conjugation to transferrin (Tsafack et al, J Lab Clin Med 1976; 127:574). Several conjugation or coupling procedures are possible to couple the targeting agent with other compounds, including but not limited to glutaraldehyde (Yeh & Faulk, Clin Immunol Immunopathol 1984; 32:1), disulfide coupling (Sasaki et al., Jap J Can Res 1993; 84:191) or benzoyl hydrazone coupling (Kratz et al., J Pharm Sci 1998; 87:338). The wide variety of coupling procedures allows the conjugation of a broad range of iron-chelating drugs to targeting proteins, resulting in either permanent or dissociable bonding of cytotoxic drugs with the protein molecule (Barabas et al., J Biol Chem 1992; 267:9437); Following the coupling reaction, drug-protein conjugates can be separated from uncoupled drug and free protein, preferably by using chromatographic or dialysis procedures.

Technical details of the conjugation procedure can vary, but the conjugates must be active in binding and killing experiments with cancer cells, and should not bind or kill significant numbers of normal cells. In light of these requirements, the preferred method for preparing the conjugates according to the present invention is the following process:

The synthesis of large amounts of homogeneous transferrin-doxorubicin conjugates with predetermined molecular ratios was done stoichiometrically by employing the only amino group of doxorubicin (DOX), which is at the 3′ amino position, to react with one of the two reactive groups on glutaraldehyde (GLU) by drop-wise addition of a saline solution of DOX into a saline solution of GLU containing a solvent such as DMSO or another suitable cryopreservative, to a final concentration of a 1:1 molar ratio of DOX-to-GLU. The resulting solution of DOX-GLU was stirred three hours at room temperature in the dark.

The molarities of DOX and GLU were the same in the above reaction in order to produce a final solution of DOX-GLU that contains neither free DOX nor free GLU. However, there is the possibility of free GLU in solution if one GLU reacts with two DOX to produce DOX-GLU-DOX, but this possibility is minimized by the mass action kinetics generated by drop-wise addition of monovalent DOX into the solution of bivalent GLU. The volumes of these reactants are not restricted, so large amounts of homogeneous DOX-GLU can be prepared.

The DOX-GLU was conjugated with a targeting protein by drop-wise addition to a saline solution of transferrin (TRF). The TRF can be either iron-free (apo-transferrin) or iron-saturated (holo-transferrin). The desired molar ratio of DOX to TRF was obtained by appropriately adjusting the volume of TRF. The resulting solution of TRF-GLU-DOX was stirred for 20 hours at room temperature in the dark. Unlike the reaction of DOX with GLU, the reaction of DOX-GLU with TRF is not restricted to one binding site, for the GLU component of DOX-GLU can react with any one of several epsilon-amino lysine groups in the TRF molecule.

The number of DOX molecules bound to TRF was determined by calculation For example, if the starting ratio of DOX-GLU to TRF was 7.2:1.0, the final solution of TRF-GLU-DOX would have contained 2.5 molecules of DOX per molecule of TRF. However, if the staring ratio of DOX-GLU to TRF was 4.0:1.0, the final solution of TRF-GLU-DOX would have contained 1.4 molecules of DOX per molecule of TRF. Similarly, if the staring ratio of DOX-GLU to TRF was 2.5:1.0, the final solution of TRF-GLU-DOX would have contained 0.9 molecules of DOX per molecule of TRF. In this way, large amounts of TRF-GLU-DOX with predetermined ratios of DOX-to-TRF can be provided according to the need.

Further steps in the conjugation reaction were the addition of ethanolamine or another substance suitable for scavenging any excess linker, followed by centrifugation and dialysis. Although reactions with DOX and TRF theoretically consume all of the GLU, ethanolamine was added to the final reaction mixture to bind any available GLU. This reaction was allowed to continue for 30 minutes in the dark The final solution was centrifuged at 2000 rpm for 10 minutes, dialyzed twice for 6 hours in a 100-fold excess of saline and three times in the same excess of Hepes buffered saline, and the resulting TRF-GLU-DOX conjugates were ready for use.

Biochemical Characterization of the Conjugates

By using HPLC and polyacrylamide gel electrophoresis as described in (39), the homogeneity of TRF-GLU-DOX conjugates can be determined. Also, by using spectrophotometry as described in (89), the molecular ratio of DOX-to-TRF can be determined. These techniques repeatedly have revealed a consistent homogeneity of the TRF-GLU-DOX conjugates. In addition, chromatography is not required in the preparation of these conjugates, because there are no aggregates or fragments. This allows for the preparation of large volumes of homogeneous transferrin-drug conjugates, which increases yields and decreases costs.

The expenses caused by losses of TRF and DOX in other types of transferrin-drug conjugates have been an impediment to their use. For example, yields of DOX and TRF are decreased by using procedures such as thiolation (44) that alter the drug and/or protein Yields also are decreased by using solvent systems (86) and by chromatography used to prepare acid-stable and acid-labile linkages (41). The GLU bond between DOX and TRF is acid-stable (89), and yields of DOX and TRF in TRF-DOX conjugates prepared according to this invention are high. Indeed, compared to other procedures (38, 39, 40), the yield for TRF is nearly doubled (90% vs 50%), and the yield for DOX is increased 5-fold.

None of the previously known approaches to the preparation of transferrin-doxorubicin conjugates are capable of producing large amounts of homogeneous conjugates with predetermined ratios of the number of drug molecules per molecule of transferrin. In addition, the other approaches employ chromatography to eliminate aggregates and to harvest fractions that are enriched in homogeneous conjugates. These procedures decrease yields, increase costs, and lack the ability to predetermine molecular ratios.

Another procedure would be to mix one milliliter of transferrin (0.5 mM) with one milliliter of deferoxamine (8.5 mM) in 150 mM sodium chloride for 4 minutes, and then add one milliliter of 21.5 mM glutaraldehyde in 150 mM sodium chloride and mix 4 minutes. The preceding reaction is a coupling procedure, which is stopped by the addition of 0.8 milliliters of 37.2 mM ethanolamine in 150 mM sodium chloride and 10 mM Hepes buffer (pH8) and vortexed for 4 minutes. The mixture (3.8 milers) then is transferred to dialysis tubing (molecular weight cutoff of 12,000–14,000), and dialyzed against 0.5 liters of Hepes-buffered saline in the dark at 5° C. for 3 hours. The dialysis should be repeated at least once with fresh Hepes-buffered saline. The mixture then is centrifuged at 1600 g for 10 minutes at 4° C. and the supernatant is chromatographed at a flow rate of 22 milliliters per hour on a 2.6×34 cm column of Sepharose CL-4B, previously equilibrated in Hepes-buffered saline and calibrated at 5° C. with blue dexran, transferrin and cytochrome C. Elution from the column is monitored at 280 nm, and 3.8 milliliter fractions are collected. The concentration of transferrin and deferoxamine in each fraction is calculated by successive approximation from standard curves from transferrin and deferoxamine, determined by using 280 nm for transferrin and 356 nm for deferoxamine. With minor modifications, this coupling procedure can be used to prepare targeting protein conjugates of other iron chelating drugs, such as protein conjugates of hydrophobic reversed siderophores.

Characterizing the Conjugates

After the pure drug-protein conjugates are isolated, they are characterized by polyacrylamide gel electrophoresis to determine their molecular weight, and the number of drug molecules per protein molecule is determined. The exact number of drug molecules per transferrin molecule can be determined, using any suitable technique including but not limited to spectrophotometric techniques. A functional drug:protein ratio is between about 0.1:1.0 to 3.0:1.0 (Berczi et al., Arch Biochem Biophys 1993; 300:356). The conjugates are checked to determine if they bind to receptors on the surface of tumor cells, and to determine if the conjugates kill cancer cells but not normal cells. Only conjugates that bind to cancer cells and not to normal cells are selected for toxicity tests using drug-sensitive and drug-resistant cancer cells. The binding studies can be done by using flow cytometry or any other suitable method, and the killing studies can be done by using microculture techniques to determine the concentration of free drug required to kill 50% of a culture of cancer cells compared to the concentration of drug in the drug-protein conjugates required to kill the same number of cancer cells. When testing the heat sensitizer conjugates, the toxicity test is done by using the MIT tetrazolium colorimetric assay (Visitica et al., Cancer Res 1994; 51:2515). These toxicity tests determine the most potent transferrin sensitizer ratio and the optimum concentration of conjugate for maximum heat sensitization of drug sensitive and drug resistant cells. Approximately 10-fold more free drug compared to drug in the drug-protein conjugate is required to kill the same number of cells.

While the above description refers to transferrin as being the delivery protein, it is known that other proteins exist in the body which are capable of binding to receptor sites on cells. If such a receptor site is activated in cancer cells and is inactive in normal cells, then any protein-or other molecule (ie., ligand) that binds to such a receptor site can be used to deliver the drugs used in the present invention. An example of such a binding protein is transcobalamin, which delivers vitamin B12 to transcobalamin receptors on cells, including cancer cells (Seetheram, Ann Rev Nutr 1999; 19:173). Low density lipoprotein is another ligand that has been conjugated to the photosensitizer chlorin and targeted to low density lipoprotein receptors on retinoblastoma cells (Schmidt-Erfurth et al., Brit J Surg 1997; 75:54).

After the drug-protein conjugate has been prepared, purified, characterized and validated for cellular binding and killing properties, and, when the binding and killing experiments show that the conjugate binds to and kills cancer but not normal cells, the conjugate is then aliquoted and sterilized. The sterilization process can be done by any suitable method including but not limited to exposure to irradiation, such as by using a cesium irradiator, or by using Millipore filtration techniques.

According to a further aspect of the present invention, there is provided a reagent kit for the treatment of tumors, comprising iron-bearing transferrin and a conjugate of transferrin with either a photosensitizer, a heat sensitizer agent or an apoptosis inducing compound. The patient's normal cells which have transferrin receptors may be protected against the effects of the conjugate by saturating these receptors with the iron-bearing transferrin before administration of the photosensitizer, heat sensitizer or apoptosis inducing conjugates.

The present invention also provides a process for determining the susceptibility of tumor cells to anti-tumor agents, comprising administering separately to portions of said tumor cells conjugates of transferrin with a number of different photosensitizer, heat sensitizer and/or apoptosis inducing agents. A reagent kit comprising a number of such different conjugates may be provided for this purpose. It has been found that tumor cells take up the conjugates of the present invention extremely rapidly. This means that within a matter of hours of removal from the patient, tumor cells may be tested against a range of conjugates of transferrin with different anti-tumor agents. Such a process would enable the chemotherapy which is most effective for a given patient to be determined as soon as possible after isolation of the tumor cells.

The conjugates according to the present invention are administered to an animal in an effective amount. In treating cancer, an effective amount includes an amount effective to: reduce the size of a tumor; slow the growth of a tumor; prevent or inhibit metastases; or increase the life expectancy of the affected animal. The present invention provides for a method of treating cancers wherein the cancer can be but is not limited to a leukemia, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, bladder cancer, gastrointestinal cancer, nasopharyngeal cancer, cervical cancer, sarcoma, myeloma, lymphoma/melanoma, glioma, or astrocytoma. The dosage for the conjugates can be determined taking into account the age, weight and condition of the patient and the pharmacokinetics of the anti-tumor agent. The amount of the conjugate required for effective treatment will be less than the amount required using the anti-tumor agent alone.

The pharmaceutical compositions of the invention can be administered by a number of routes, including but not limited to orally, topically, rectally, vaginally, by pulmonary route, for instance, by use of an aerosol, or parenterally, including but not limited to intramuscularly, subcutaneously, intraperitoneally, intra-arterially or intravenously. The compositions can be administered alone, or can be combined with a pharmaceutically-acceptable carrier or excipient according to standard pharmaceutical practice. For the oral mode of administration, the compositions can be used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. For parenteral administration, sterile solutions of the conjugate are usually prepared, and the pHs of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular adminstration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. For pulmonary administration, diluents and/or carriers will be selected to be appropriate to allow the formation of an aerosol. It is preferred that the conjugate of the present invention be administered parenterally, i.e. intravenously or intraperitoneally, by infusion or injection.

Preferred embodiments of the present invention are described below. It will be apparent to those of ordinary skill in the art after reading the following description that modifications and variations are possible, all of which are intended to fall within the scope of the claims.

EXAMPLE 1

Preparation of a Homogeneous Transferrin-Doxorubicin Conjugate

The synthesis of large amounts of homogeneous transferrin-doxorubicin conjugates with predetermined molecular ratios was done stoichiometrically by employing the only amino group of doxorubicin (DOX), which is at the 3′ amino position, to react with one of the two reactive groups on glutaraldehyde (GLU). The first step was to add GLU drop-wise to DMSO in an ice cold water bath. Next was the drop-wise addition of a saline solution of DOX into a saline solution of GLU+DMSO to a final concentration of a 1:1 molar ratio of DOX-to-GLU. The resulting solution of DOX-GLU was stirred three hours at room temperature in the dark.

The molarities of DOX and GLU were the same in the above reaction in order to produce a final solution of DOX-GLU that contains neither free DOX nor free GLU. However, there is the possibility of free GLU in solution if one GLU reacts with two DOX to produce DOX-GLU-DOX, but this possibility is minimized by the mass action kinetics generated by drop-wise addition of monovalent DOX into the solution of bivalent GLU. The volumes of these reactants are not restricted, so large amounts of homogeneous DOX-GLU can be prepared.

Subsequently DOX-GLU was added to transferrin (TRF) by drop-wise addition into a saline solution of the transferrin (TRF). The TRF can be either iron-free (apo-transferrin) or iron-saturated (holo-transfernin). The desired molar ratio of DOX to TRF was obtained by appropriately adjusting the volume of TRF. The resulting solution of TRF-GLU-DOX was stirred for 20 hours at room temperature in the dark Unlike the reaction of DOX with GLU, the reaction of DOX-GLU with TRF is not restricted to one binding site, for the GLU component of DOX-GLU can react with any one of several epsilon-amino lysine groups in the TRF molecule.

The number of DOX molecules bound to TRF was determined in the second step. For example, if the starting ratio of DOX-GLU to TRF was 7.2:1.0, the final solution of TRF-GLU-DOX would have contained 2.5 molecules of DOX per molecule of TRF. However, if the starting ratio of DOX-GLU to TRF was 4.0:1.0, the final solution of TRF-GLU-DOX would have contained 1.4 molecules of DOX per molecule of TRF. Similarly, if the starting ratio of DOX-GLU to TRF was 2.5:1.0, the final solution of TRF-GLU-DOX would have contained 0.9 molecules of DOX per molecule of TRF. In this way, large amounts of TRF-GLU-DOX with predetermined ratios of DOX-to-TRF can be provided according to the need.

Any excess GLU remaining in the reaction product can be scavenged by the addition of ethanolamine, followed by centrifigation and dialysis. Although reactions with DOX and TRF theoretically consume all of the GLU, ethanolamine was added to the final reaction mixture to bind any available GLU. This reaction was allowed to continue for 30 minutes in the dark. The final solution was centrifuged at 2000 rpm for 10 minutes, dialyzed twice for 6 hours in a 100-fold excess of saline and three times in the same excess of Hepes buffered saline, and the resulting TRF-GLU-DOX conjugates were ready for use.

Biochemical Characterization of the Conjugates

By using HPLC and polyacrylamide gel electrophoresis, the homogeneity of TRF-GLU-DOX conjugates can be determined Also, by using spectrophotometry, the molecular ratio of DOX-to-TRF can be determined. These techniques repeatedly have revealed a consistent homogeneity of the TRF-GLU-DOX conjugates. In addition, chromatography is not required in the preparation of these conjugates, because there are no aggregates or fragments. This allows for the preparation of large volumes of homogeneous transferrin-drug conjugates, which increases yields and decreases costs.

The expenses caused by losses of TRF and DOX in other types of transferrin-drug conjugates have been an impediment to their use. For example, yields of DOX and TRF are decreased by using procedures such as thiolation that alter the drug and/or protein. Yields also are decreased by using solvent systems and by chromatography used to prepare acid-stable and acid-labile linkages. The GLU bond between DOX and TRF is acid-stable, and yields of DOX and TRF in TRF-DOX conjugates prepared according to this invention are high Indeed, compared to other known procedures, the yield for TRF is nearly doubled (90% vs 50%), and the yield for DOX is increased 5-fold.

None of the previously known approaches to the preparation of transferrin-doxorubicin conjugates are capable of producing large amounts of homogeneous conjugates with predetermined ratios of the number of drug molecules per molecule of transferring. In addition, the other approaches employ chromatography to eliminate aggregates and to harvest fractions that are enriched in homogeneous conjugates. These procedures decrease yields, increase costs, and lack the ability to predetermine molecular ratios.

EXAMPLE 2

Preparation of a Transferrin-Apoptosis Inducing Compound (Deferoxamine) Conjugate

Glutaraldehyde (GLU) is added drop-wise to DMSO in an ice cold water bath followed by the drop-wise addition of a saline solution of deferoxamine (DEF) into a saline solution of GLU+DMSO to a final concentration of a 2:1 molar ratio of DEF-to-GLU. The resulting solution of DEF-GLU is stirred three hours at room temperature in the dark.

The DEF-GLU is added drop-wise to a saline solution of transferrin (TRF). The TRF can be either iron-free (apo-transferrin) or iron-saturated (holo-transferrin). The desired molar ratio of DEF to TRF is obtained by appropriately adjusting the volume of TRF. The resulting solution of TRF-GLU-DEF was stirred for 20 hours at room temperature in the dark. Unlike the reaction of DEF with GLU, the reaction of DEF-GLU with TRE is not restricted to one binding site, for the GLU component of DEF-GLU can react with any one of several epsilon-amino lysine groups in the TRF molecule.

The number of DEF molecules bound to TRF is determined during the addition of DEF-GLU to TRF. For example, if the starting ratio of DEF-GLU to TRF was 7.2:1.0, the final solution of TRF-GLU-DEF would have contained 2.5 molecules of DEF per molecule of TRF. However, if the starting ratio of DEF-GLU to TRF is 4.0:1.0, the final solution of TRF-GLU-DEF would have contain 1.4 molecules of DEF per molecule of TRF. Similarly, if the starting ratio of DEF-GLU to TRF is 2.5:1.0, the final solution of TRF-GLU-DEF would contain 0.9 molecules of DEF per molecule of TRF. In this way, large amounts of TRF-GLU-DEF with predetermined ratios of DEF-to-TRF can be provided according to need.

Ethanolamine is normally added to the reaction product followed by centrifugation and dialysis. Although reactions with DEF and TRF theoretically consume all of the GLU, ethanolamine is added to the final reaction mixture to bind any available GLU. This reaction is allowed to continue for 30 minutes in the dark. The final solution is centrifuged at 2000 rpm for 10 minutes, dialyzed twice for 6 hours in a 100-fold excess of saline and three times in the same excess of Hepes buffered saline, and the resulting TRF-GLU-DEF conjugates are ready for use.

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1. A homogeneous conjugate comprising a targeting agent linked to a bioaffecting material via a glutaraldehyde linker, wherein said bioaffecting material is doxorubicin and said targeting agent is transferrin, wherein said homogeneous conjugate is free of dimers, trimers and aggregates and wherein said conjugate has a doxorubicin: transferrin molar ratio of about 2:1.
 2. The homogeneous conjugate according to claim 1, wherein said conjugate is made by a process comprising: a) adding a solution of doxorubicin dropwise to a molar equivalent of a glutaraldehyde solution to link one doxorubicin molecule to one glutaraldehyde molecule in a doxorubicin/glutaraldehyde combination; and b) adding the doxorubicin/glutaraldehyde combination to transferrin to produce said homogeneous conjugate.
 3. A kit for treating tumors, comprising in separate containers (a) a conjugate according to claim 1, and (b) a second conjugate comprising a targeting agent and a bioaffecting material, wherein said bioaffecting material is selected from the group consisting of a heat sensitizer, a photosensitizer, and an apoptosis inducing compound and said targeting agent is selected from the group consisting of transferrin, somatostatin, epidermal growth factor, folic acid and transcobalamin. 